Keywords Nonylphenol - Hsp27 - locomotion - survival -
Drosophila
Introduction
Nonylphenol is an endocrine-disrupting chemical (EDC) and a persistent alkyl phenolic compound, which is widely used in both industrial and domestic as a surfactant, paints, plastics, herbicides, and pesticide.[1 ] According to the Environmental Protection Agency (EPA) recommendations, the permissible limit of nonylphenol is 1.7 μg/L in salt water and 6.6 μg/L in freshwater.[2 ] However, the nonylphenol concentrations of 1.22 to 7.24 μg/L, 3.31 to 30.96 μg/kg, and 18.03 to 23.89 mg/kg have been recorded in water, sediments, and tomato plant, respectively.[3 ]
[4 ] Living organisms are exposed to nonylphenol throughout their lives via the ingestion of contaminated food/water, causing a disturbance in hormonal balance and consequently leading to various health disorders such as behavior disorders, developmental and reproductive abnormalities, and alterations in immune functions.[5 ]
[6 ]
[7 ] In a study by Paolella et al,[8 ] nonylphenol exposure causes ER stress, apoptosis, and mitochondrial dysfunction in the hepatic cell line. Additionally, it was noted that nonylphenol elicits oxidative stress in a dose- and concentration dependent manner. The study also revealed that when exposed to nonylphenol, the Keap1–Nrf2 pathway was activated, reducing the damage caused by oxidative stress at lower doses and shorter exposure. However, at higher doses and chronic exposure, the Keap1–Nrf2 pathway is inhibited, thereby increasing oxidative stress.[9 ] An organism's gut interfaces between environmental conditions and an individual's fitness. In this line, several studies have underlined toxicants, such as microplastic and pesticide-induced gut toxicity may lead to various health adversities.[10 ]
[11 ] Similarly, our recently published study shows that nonylphenol exposure to Drosophila larvae leads to oxidative stress in the gut, which is further associated with poor developmental indices.[12 ] Therefore, restoring or maintaining gut health might help preserve an organism's fitness against nonylphenol exposure.
The cellular machinery is orchestrated to execute the various defense responses against different stress types, including chemical stress.[13 ] In this line, the induction of heat shock protein (Hsp) has been established as the primary cellular defense mechanism. Hsp27 is a member of the small Hsp family and is known for its anti-aging and anti-oxidative nature.[14 ] Our previous results have shown that Hsp27 overexpression in the midgut of Drosophila larvae confers cellular protection against nonylphenol via decreasing oxidative stress.[12 ] Therefore, in continuation of our previous work, this study aims to investigate the role of Hsp27 in the midgut against nonylphenol-induced locomotory and survival toxicity using the Drosophila melanogaster model system.
Drosophila is widely used as an alternative animal model for toxicological studies.[15 ] Like higher vertebrates, Drosophila also exhibits an age-dependent decline in survival and behavioral function. Locomotory behavior is a reliable parameter to assess motor function in flies. The survival of an organism depicts the overall health of the organism. These characteristics can be affected by an environmental factor or disease etiology, leading to the progression of aging and life-shortening effects. Typically, the life span of healthy Drosophila is approximately 65 to 70 days at 25°C.[16 ] Moreover, Drosophila has been previously used to understand the link between Hsp27 , aging, and locomotory behavior.[17 ] The European Centre for the Validation of Alternative Methods (ECVAM) recommends Drosophila as an alternative model that follows the 3R rule, i.e., reducing, refining, and replacing the use of laboratory animals.[18 ]
Methods and Methodology
Experimental Organisms and Culture
In the study, the stock w1118
and Gal4-UAS transgenic lines of Drosophila melanogaster , specifically Np1-Gal4 (GAL4 expressed in the Drosophila midgut) and UAS-Hsp27 (expressed Hsp27 under the control of UAS) ([Fig. 1 ]). Drosophila strains were reared on standard media containing agar-agar, maize powder, sugar, yeast, nipagin, and propionic acid at 24°C with 12 hour light/dark cycles. An additional yeast supplement was given to ensure the organisms' proper growth.
Fig. 1 Schematic representation showing the crossing scheme between Np1-Gal4 and UAS-Hsp27 .
Exposure Regime
The study used a nonylphenol concentration of 5.0 µg/mL (Sigma-Aldrich). Nonylphenol concentrations were selected based on the previous study.[12 ]
[19 ] Flies were transferred into nonylphenol mixed food (dissolved in 0.3% dimethyl sulfoxide [DMSO]; final concentration). For control and vehicle control, flies were fed a conventional diet and the diet containing 0.3% DMSO, respectively.
Locomotory Assay
The locomotory behavior of the organism was assessed according to the previously described method with a few modifications.[20 ] In brief, the newly emerged flies (after 2–3 days post eclosion) were subjected to nonylphenol for 10 to 40 days, and the climbing activity of control and nonylphenol-exposed flies was assessed after each 10 days exposure interval. In brief, the flies were placed in a locomotory chamber marked 15 cm (20 flies/vial). The flies were gently tapped down to the bottom of the locomotory chamber and allowed to move toward the 15 cm mark. The number of flies that crossed the 15 cm mark in 30 seconds in the locomotory chamber was documented. The climbing activity was determined as the average number of flies climbed 15 cm in three replicates (three trials/replicate) and represented as % locomotory performance.
Survival Assay
The newly emerged Drosophila were used to assess the effect of nonylphenol on survivability. The organisms (50 flies/vial and 5 vials/group) were transferred to the normal food and food-containing nonylphenol from the first day of their eclosion to 40 days. The control and nonylphenol-exposed flies were transferred to the new respective diet every alternate day, and the mortality was recorded in each vial for 40 days.
Statistical Analysis
The Prism software (GraphPad version 8.4, San Diego, CA, USA) was used for statistical analysis. The analysis of variance (ANOVA) was used to determine the statistical significance in control and treated flies. The p -values were ascribed as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). While the comparison with overexpressed Hsp27 flies to wild-type flies, the significance is represented as p < 0.001 (
$$$
).
Results
The experiments were performed using Drosophila adult flies, and the overexpression of Hsp27 in the Drosophila gut was achieved through a standard genetic cross between Np1-gal4 and UAS-Hsp27 strains. The study found no overt harmful effects of nonylphenol exposure in Drosophila flies. Drosophila flies fed with 0.3% DMSO (vehicle control) showed no significant change. Therefore, the data presented in the study were compared against the biological control.
Hsp27 Overexpression in the Drosophila Gut Resists the Nonylphenol-induced Locomotory Decline
To examine the Hsp27 role in the locomotory behavior of the organism, we have assessed the climbing activity of nonylphenol-exposed flies. An insignificant change was observed in the climbing activity of Np1-Gal4 > w1118,
and Np1-Gal4 > UAS-Hsp27 flies after 10 to 20 days of nonylphenol exposure. However, a significant reduction in the climbing activity was observed after 30 (∼20%, p < 0.01 ) and 40 (∼76%, p < 0.001 ) days of nonylphenol exposure in Np1-Gal4> w1118
as compared with the control ([Fig. 2 ]). While Np1-Gal4 > UAS-Hsp27 showed a comparatively lesser reduction of ∼13% (30 days) to ∼23% (40 days) in the climbing performance in exposed flies as compared with Np1-Gal4 > UAS-Hsp27 flies. Moreover, Np1-Gal4 > UAS-Hsp27 flies exposed to 5.0 µg/mL nonylphenol showed ̴ 35% to ̴ 69% improvement climbing than flies Np1-Gal4 > w1118
to 30 and 40 days nonylphenol exposure, respectively ([Fig. 2 ]).
Fig. 2 Overexpression of Hsp27 improves the locomotory performance in nonylphenol-exposed adult Drosophila emerged. Significance is ascribed to p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). While the comparison with overexpressed Np1-Gal4 > UAS-Hsp27 flies to Np1-Gal4 > w1118
, the significance is represented as p < 0.001 (
$$$
). Data represent mean ± SD (n = 3).
Hsp27 Overexpression Improves the Survivability of the Nonylphenol-exposed Organism
To analyze the impact of Hsp27 overexpression in the organism's gut on longevity, a survival assay was performed in nonylphenol-exposed flies. An insignificant change was observed in the survival of flies until 25 days of nonylphenol exposure in Np1-Gal4 > w1118
and Np1-Gal4 > UAS-Hsp27 ([Fig. 3 ]). A significant (p < 0.001 ) reduction in survival was observed after 35 to 40 days of nonylphenol exposure in Np1-Gal4 > w1118
compared with the control. The maximum decrease in survival of the organism was observed after 40 days (∼35%) of nonylphenol exposure in Np1-Gal4> w1118 . Under similar exposure conditions Np1-Gal4 > UAS-Hsp27, flies displayed a reduction of ∼20% after nonylphenol exposure compared with the respective control flies ([Fig. 3 ]). Altogether Np1-Gal4 > UAS-Hsp27 flies showed ∼40% survival benefit compared with Np1-Gal4 > w1118
after nonylphenol exposure.
Fig. 3
Hsp27 overexpression diminishes the nonylphenol-induced survival toxicity in adult Drosophila . The data are represented in the form of percentage survival. Significance is ascribed to p < 0.01 (**), and p < 0.001 (***). While the comparison with overexpressed Np1-Gal4 > UAS-Hsp27 flies to Np1-Gal4 > w1118
, the significance is represented as p < 0.001 (
$$$
). Data represent mean ± SD (n = 5).
Discussion
The gut is the primary organ for ingestion/oral exposure. Several first-line defense mechanisms, such as induction of stress proteins, activation of metabolic reactions, and tissue repair process, are orchestrated in the gut in response to biological, physical, and chemical stress, which eventually provide the organism's short- or long-term protection.[21 ]
[22 ]
[23 ] In addition, the gut harbors microorganisms, which play a critical role in regulating several organisms' responses/physiological processes, such as immune response, xenobiotics metabolism, digestion, and absorption.[24 ]
[25 ]
Nonylphenol enters the organism's system mainly through water and food. It readily gets absorbed in an organism's gut and affects gut homeostasis.[26 ] Our previous study revealed that exposure to nonylphenol via food hampers the emergence pattern and causes cellular adversities such as increased oxidative stress and cell death in the midgut Drosophila larvae.[12 ] However, modulation of Hsp27 by overexpressing in Drosophila larvae midgut improves the emergences pattern and reduces cellular toxicity. In the study, we aimed to explore the role of Hsp27 overexpression in the gut against nonylphenol-caused locomotory and survival toxicity in exposed adult Drosophila.
Overexpression of Hsp27 Diminishes the Locomotory Toxicity due to Nonylphenol Exposure
The gut is the second brain of the organism, as it contains a series of enteric nervous system which runs from the esophagus, through the stomach and intestines to the anus. This enteric nervous system depends on the same kind of neurons and neurotransmitters found in the brain. The neuronal cross-talk between the gut and brain is known as the gut–brain axis. Prior studies have shown that a disturbed gut is linked with mental health.[27 ] The disturbing gut homeostasis causes an increase in the inflammation and the permeability of the gut barrier, which hampers the blood–brain barriers and promotes the progression of neurodegeneration diseases. Prior study has shown that exposure to bisphenol A hampers the tight intestinal junctions, leading to gut barrier dysfunction. Its exposure also decreases the level of serum neurotransmitters, affects the hippocampus, and tryptophan, 5-hydroxytryptamine (5-HT), and 5-hydroxy indole acetic acid and microbial community, which later promotes impaired mental capabilities and inflammation in the intestine and brain.[28 ] Therefore, restoring gut health could be the primary way to improve neurological issues. Hsp27 functions as an antioxidant by lowering reactive oxygen species (ROS) and increasing the intracellular glutathione.[12 ] In this aspect, our study has shown that overexpression of Hsp27 (Np1-Gal4 > UAS-Hsp27 ) rescued locomotor insufficiency on exposure to nonylphenol. With this, previous research has also demonstrated the protective role of Hsp27 in different organ systems. For example, previous studies have shown that overexpression of Hsp27 in the brain ameliorates symptoms of neurological diseases.[17 ]
[29 ]
Hsp27 Overexpression Improves the Nonylphenol-Induced Survival Toxicity
The gut has a permeable wall that restricts the passage of bacteria and harmful chemicals while permitting nutrients and immunological signaling. The dysfunction in the gut causes leaking of the gut, inflammation, and premature aging. Prior study has shown that dietary restriction improves intestinal integrity by modulating dMyc and decreasing cell death, increasing the organisms' life span.[30 ] Similarly, upregulation in the Hsp27 has been shown to inhibit apoptosis.[14 ] Our previous study demonstrated that Hsp27 overexpression in the midgut of Drosophila larvae reduces oxidative stress and cell death and improves host fitness upon nonylphenol exposure.[12 ] In the present study, we have shown a significant improvement in life span in nonylphenol-exposed Np1-Gal4 > UAS-Hsp27 flies compared with Np1-Gal4> w1118
flies. Alexander et al[31 ] showed that Hsp27 (HspB1) improves the life span of the organisms by decreasing the oxidative stress in exposed Caenorhabditis elegans.
Considering the foregoing, the overexpression of Hsp27 in the gut of adult Drosophila exposed to nonylphenol improves the life span and locomotory behavior of exposed Drosophila ([Fig. 4 ]). The overexpression of this chaperone might decreases oxidative stress due to nonylphenol exposure. This focuses on therapeutic attention as the target moiety toward organismal (including human) health in xenobiotic-contaminated environments. Due to the genetic and functional homology between Drosophila and mammalians, the resultant information generated in this study aids in understanding the nonylphenol-induced toxicity in a higher organism.
Fig. 4 Schematic representation of the protective function of Hsp27 against prolonged nonylphenol exposure-induced locomotory and survival toxicity.
Conclusion
In conclusion, the present study indicates that nonylphenol 5.0 µg/mL exposure to adult Drosophila induces deficits in locomotory activity and lifespan. We showed that prolonged exposure to nonylphenol causes a significant reduction in climbing activity and survival after 30 to 40 days of exposure. Interestingly, Hsp27 overexpression in the midgut of Drosophila reverses the impact of nonylphenol-induced locomotory and survivability toxicity, which highlights the role of gut health in organisms' fitness. The study shows that because higher vertebrates and Drosophila share many genetic and functional characteristics, Hsp27 may represent a significant therapeutic target against nonylphenol exposure.
